microRNA-26a represses pancreatic cancer cell malignant behaviors by targeting E2F7

Abstract

Dysregulation of microRNAs (miRNAs) exerts key roles in the development of pancreatic cancer (PCa). miR-26a is reportedly a tumor suppressor in cancers. However, whether miR-26a modulates PCa progression is poorly understood. Here, we found that miR-26a was down-regulated in PCa. Overexpressed miR-26a suppressed PCa cell proliferation, colony formation, and tumor stem cell properties. Mechanically, the transcription factor E2F7 is a downstream target of miR-26a. miR-26a decreased E2F7 expression through binding to the 3'-untranslated region (UTR) of E2F7. Decreased miR-26a in PCa tissues was inversely correlated with E2F7. The inhibitory effects of miR-26a in PCa were reversed by E2F7 overexpression. Consistently, the knockout of E2F7 further significantly inhibited the growth of PCa cells combined with miR-26a overexpression. Further study revealed that E2F7 bound the promoter of vascular endothelial growth factor A (VEGFA), a key factor in angiogenesis, and transcriptionally activated the expression of VEGFA. miR-26a overexpression attenuated the effects of E2F7 on VEGFA promotion. Our results uncovered the novel function of miR-26a/E2F7/VEGFA in PCa, making miR-26a a possible target for PCa treatment.

Keywords: Cell proliferation; E2F7; Pancreatic cancer; VEGFA; miR-26a.

Fig. 1

miR-26a expression was down-regulated in PCa. A A comparison of miR-26a expression in 50 paired PCa tissues and adjacent non-cancer tissues. B miR-26a levels in normal HPDE-C7 cell and PCa cell lines. Sw1990, PANC-1, AsPC-1, and BXPC-3. *P < 0.05; ***P < 0.001

Fig. 2

miR-26a suppressed the proliferative ability of PCa cells. A miR-26a expression of AsPC-1 and PANC-1 cells after miR-26a transfection. B, C The proliferation of PCa cells with overexpression of miR-26a was significantly inhibited. D miR-26a transfection decreased the number of PCa cells. E miR-26a enhanced apoptosis of both AsPC-1 and PANC-1 cells. F The expression levels of the stemness markers in PCa cells were examined after transfection of miR-26a. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

miR-26a targeted E2F7 in PCa. A The possible binding site of miR-26a in the 3’-UTR of E2F7 mRNA. B, C The luciferase activity of AsPC-1 and PANC-1 cells that were co-transfected with miR-26a and either vector expressing WT or Mut 3’-UTR of E2F7. D, E We detected the mRNA or protein expression of E2F7 in PCa cells that were overexpressed with miR-26a. ***P < 0.001

Fig. 4

Overexpressed E2F7 in PCa attenuated the suppressive role of miR-26a. A E2F7 mRNA levels in the indicated tissues samples were detected using RT-qPCR. The representative IHC staining of VEGFA in PCa tissues and adjacent normal tissues. Scale bar, 50 um. B The protein levels of E2F7 in normal and PCa cells were compared by Western blot. C Spearman correlation test showed the inverse correlation between miR-26a and E2F7 in PCa. D We transfected PCa cells with the indicated vectors and detected E2F7 expression. E, F Reintroduction of E2F7 significantly reversed the suppressed proliferation by miR-26a. G The endogenous level of E2F7 was knocked out by Caspir-cas9, and the silencing efficiency was confirmed via Western blotting. H, I Silencing of E2F7 expression further inhibited PCa cell proliferation by miR-26a overexpression. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5

miR-26a negatively regulated E2F7-mediated transcriptional activation of VEGFA. A mRNA levels of VEGFA were overexpressed in PCa tissues. B, C The binding between E2F7 with the VEGFA promoter under different conditions was detected by CHIP. D The binding of E2F7 with the VEGFA promoter region was examined by luciferase reporter analysis. E The mRNA levels of VEGFA under the indicated conditions were compared via RT-qPCR. F, G The correlation between the expression of VEGFA with miR-26a or E2F7 in PCa tissues. ***P < 0.001